The described invention relates in general to methods for studying biological systems, and more specifically to a system and method for isolating a single mitochondrion isolated from a eukaryotic cell for the primary purpose of identifying one or more specific mitochondrial heteroplasmies in the cell from which the mitochondria was obtained.
In cell biology, a mitochondrion (plural mitochondria) is a membrane-enclosed organelle found in most eukaryotic cells. These organelles are typically about 1-10 μm in size and are often described as “cellular power plants” because they generate most of the cell's supply of adenosine triphosphate (ATP), which is used as a source of chemical energy. In addition to supplying cellular energy, mitochondria are involved in a range of other processes, including signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth. Mitochondria have been implicated in several human diseases and may also play a role in the aging process. The number of mitochondria in a cell varies widely by organism and tissue type. Some cells may have only a single mitochondrion or very few mitochondria, whereas others can contain several thousand mitochondria. In humans, the mitochondria may contain about 615 distinct proteins, depending on the tissue of origin. Although most of a cell's DNA is contained in the cell nucleus, a mitochondrion has its own independent genome.
The human mitochondrial genome is a circular DNA molecule of about 16 kilobases. It encodes 37 genes: 13 for subunits of respiratory complexes I, III, IV, and V, 22 for mitochondrial tRNA, and 2 for rRNA. One mitochondrion can contain a variable number of copies of its DNA. As in prokaryotes, there is a very high proportion of coding DNA and an absence of repeats. Mitochondrial genes are transcribed as multigenic transcripts, which are cleaved and polyadenylated to yield mature mRNAs. Not all proteins necessary for mitochondrial function are encoded by the mitochondrial genome; most are coded by genes in the cell nucleus and imported to the mitochondrion. Thus, a mitochondrial disorder can be secondary to a mutation in either the nuclear DNA or in the mitochondrial DNA. The exact number of genes encoded by the nucleus and the mitochondrial genome differs between species.
The entire human mitochondrial DNA (mtDNA) sequence has been determined. See, e.g., Anderson, et al., “Sequence and organization of the human mitochondrial genome”, Nature 290, 457 (1981); Andrews, et al., and “Reanalysis and revision of the Cambridge Reference Sequence for human mitochodrial DNA”, Nature Genetics 23, 147 (1999). Mitochondrial genetics differ from nuclear (standard or Mendelian) genetics. Virtually all the mtDNA of a zygote is derived from the oocyte, and mtDNA disorders are transmitted by maternal inheritance. Maternal-linked (matrilineal) relatives presumably have identical mtDNA sequences, except perhaps at the site of a new mutation. Additionally, the mtDNA mutation rate is substantially higher than that of the nuclear DNA. Most cells contain dozens to thousands of mitochondria, and each mitochondrion contains several copies of mtDNA, resulting in high mtDNA copy number.
A wide variety of clinical manifestations are due to mutations in mitochondrial DNA, but are difficult to diagnose due to the varied clinical picture and the lack of sensitive or specific diagnostic testing. Past efforts to document mtDNA mutations in children believed to have mitochondrial disorders have been hampered by the size of the mitochondrial genome and the presence of numerous benign polymorphisms. Mitochondrial mutations can be single point mutations, or larger mutations deletions, insertions, rearrangements or duplications). Clinical mitochondrial dysfunction may be defined as idiopathic neuromuscular or multisystem disease, biochemical signs of energy depletion, and lack of another diagnosis. Mitochondrial disorders are evidenced when the cellular supply of energy is unable to keep up with demand and symptoms predominate in tissues with the highest energy requirements, such as brain and muscle. Mitochondrial disorders are most commonly displayed as neuromuscular disorders, including developmental delay, seizure disorders, hypotonia, skeletal muscle weakness and cardiomyopathy. Other manifestations which have been reported include gastroesophageal reflux, apnea, optic atrophy, deafness, acute liver failure, diabetes mellitus, and other hormonal deficiencies. Disorders such as MELAS (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes), MERRF (myoclonic epilepsy with ragged-red fibers) and LHON (Leber's hereditary optic neuropathy) all result from single point mutations in the mtDNA genome.
Mammalian mitochondrial DNA (mtDNA) undergoes mutations at a higher rate than nuclear DNA. If all the mtDNA has the same mutation, the DNA is called homoplasmic; but a mixture of wild type and mutation is designated heteroplasmic. Thus, heteroplasmy is defined as the presence of a mixture of more than one type of an organellar genome (mitochondrial DNA or plastid DNA) within a cell or individual. The presence and degree of heteroplasmy is important in diagnostic medicine because heteroplasmies have been linked to mitochondrial-based diseases such as those listed above, which are maternally inherited. Since eukaryotic cells can contain hundreds of mitochondria with hundreds of copies of mtDNA, it is possible for single point mutations to affect only some of mitochondria and not others, thus giving rise to heteroplasmic mitochondrial genomes within a single individual.
Mitochondrial DNA (mtDNA) heteroplasmies are well documented at the multi-cell level; however, PCR and DNA sequencing (the recognized standard for determining the presence of heteroplasmy) can typically only distinguish heteroplasmy if it is present at least in 20% of the sample. The importance of detecting low frequency mutations is demonstrated by a number of cases in which a mother may not have a detectable mutation nor exhibit any symptoms of a mtDNA disease, yet her child does have the mutation and the mitochondrial DNA disease symptoms. In such cases, it is likely that the mutated genome copy number in the mother was too low to verify the presence of a mitochondrial DNA mutation using conventional methods. Most tissue studies in a typical genetic screening begin with millions of mitochondrial DNA copies as well as nuclear DNA, which adds to the overall complexity of the sample. By reducing the sample to a single mitochondrion and eliminating all other mitochondria and nuclear DNA, the mitochondrial DNA mutations found in a single mitochondrion will become detectable. Also, the detectable presence of heteroplasmy may differ depending on the type of tissue being examined meaning that not all tissue samples will yield consistent and reproducible results. Thus, there is a need for a sensitive and reliable method of testing a subject for risk of developing mitochondrial dysfunction or disease based on the presence or absence of mitochondrial heteroplasmy within their cells.